U.S. patent application number 13/274363 was filed with the patent office on 2012-05-10 for method of treating hepatocellular carcinoma.
This patent application is currently assigned to NIIKI PHARMA INC.. Invention is credited to Walter Berger, Petra Heffeter, Bernhard Keppler, Hooshmand Sheshbaradaran.
Application Number | 20120115833 13/274363 |
Document ID | / |
Family ID | 42982899 |
Filed Date | 2012-05-10 |
United States Patent
Application |
20120115833 |
Kind Code |
A1 |
Berger; Walter ; et
al. |
May 10, 2012 |
METHOD OF TREATING HEPATOCELLULAR CARCINOMA
Abstract
A therapeutic method for treating hepatocellular carcinoma is
disclosed comprising administering to a patient in need of
treatment a ruthenium complex salt.
Inventors: |
Berger; Walter; (Vienna,
AT) ; Heffeter; Petra; (Vienna, AT) ; Keppler;
Bernhard; (Vienna, AT) ; Sheshbaradaran;
Hooshmand; (Hoboken, NJ) |
Assignee: |
NIIKI PHARMA INC.
Hoboken
NJ
|
Family ID: |
42982899 |
Appl. No.: |
13/274363 |
Filed: |
October 17, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2010/031591 |
Apr 19, 2010 |
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13274363 |
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61170534 |
Apr 17, 2009 |
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61266926 |
Dec 4, 2009 |
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Current U.S.
Class: |
514/186 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 33/24 20130101; A61K 31/555 20130101 |
Class at
Publication: |
514/186 |
International
Class: |
A61K 31/555 20060101
A61K031/555; A61P 35/00 20060101 A61P035/00 |
Claims
1. A method of treating, or delaying the onset of, hepatocellular
carcinoma, comprising administering to a patient in need of
treatment an effective amount of a compound that is
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] or a
pharmaceutically acceptable salt thereof.
2. The method of claim 1, wherein the compound is an alkali metal
salt of trans-[tetrachlorobis(1H-indazole)ruthenate(III)].
3. The method of claim 1, wherein the compound is sodium
trans-[tetrachlorobis(1H-indazole)ruthenate(III)].
4. The method of claim 1, wherein the hepatocellular carcinoma is a
refractory or recurrent hepatocellular carcinoma.
5. The method of claim 4, wherein the hepatocellular carcinoma is
refractory or resistant to one or more agents chosen from the group
consisting of sorafenib, regorafenib, anthracyclines, platinum
agents, 5-FU or capecitabine.
6. The method of claim 4, wherein the hepatocellular carcinoma is
refractory or resistant to sorafenib.
7. The method of claim 4, wherein the hepatocellular carcinoma is
refractory or resistant to doxorubicin.
8. The method of claim 4, wherein the hepatocellular carcinoma is
refractory or resistant to oxaliplatin or cisplatin.
9. The method of claim 4, wherein the hepatocellular carcinoma is
refractory or resistant to 5-FU.
10. The method of claim 1, further comprising administering to the
patient an effective amount of an embolizing agent.
11. A method of treating hepatocellular carcinoma, comprising:
identifying a patient having hepatocellular carcinoma; and treating
the patient with a therapeutically effective amount of a
pharmaceutically acceptable salt of
trans-[tetrachlorobis(1H-indazole)ruthenate(III)].
12. A kit comprising a unit dosage form of a pharmaceutically
acceptable salt of trans-[terachlorobis(1H-indazole)ruthenate(III)]
and a unit dosage form of an embolizing agent.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application is a continuation of PCT/US2010/031591
filed on Apr. 19, 2010, which is entitled to the priority of U.S.
Provisional Application No. 61/170,534 filed on Apr. 17, 2009, and
U.S. Provisional Application No. 61/266,926 filed on Dec. 4, 2009,
the contents of all of which are incorporated by reference herein
in their entirety.
FIELD OF THE INVENTION
[0002] The present invention generally relates to methods for
treating cancer, and particularly to a method of treating
hepatocellular carcinoma.
BACKGROUND OF THE INVENTION
[0003] Primary liver cancer is one of the most common forms of
cancer in the world. There are two main types of liver cancer.
Hepatocellular carcinoma (HCC, also known as malignant hepatoma)
and cholangiocellular carcinoma. HCC is the most common form of
primary liver cancer, and develops within the hepatocyte. HCC
occurs mostly in men and patients that suffer from cirrhosis. In
contrast, cholangiocellular carcinoma or bile duct cancer develops
in the small bile ducts within the liver. This type of cancer is
more common among women.
[0004] Treatment options for hepatocellular carcinoma have been
limited, especially in the case of advanced or recurrent
hepatocellular carcinoma. Surgery and radiation therapy are options
for early stage liver cancer, but not very effective for advanced
or recurrent hepatocellular carcinoma. Systematic chemotherapies
have not been particularly effective, and there are a very limited
number of drugs available for use. The recently approved kinase
inhibitor sorafenib has been shown to be effective in treating
hepatocellular carcinoma. However, it can slow or stop advanced
liver cancer from progressing for only a few months longer than
without treatment. Indeed, in a Spanish phase III clinical trial in
late stage HCC patients with well preserved liver function, it only
added an average of two months to the lifespan.
[0005] Sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] has
been shown to be effective in killing tumor cells in colon caner
cell lines SW480 and HT29. Kapitza et al., J. Cancer Res. Clin.
Oncol., 131(2):101-10 (2005). However, it is not known whether it
would be effective in treating hepatocellular carcinoma.
SUMMARY OF THE INVENTION
[0006] It has now been discovered that the compound sodium
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is especially
effective in treating hepatocellular carcinoma. It has also been
surprisingly discovered that the compound sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] is equally
effective in hepatocellular carcinoma cell lines both sensitive and
insensitive to drugs such as sorafenib, doxorubicin, cisplatin,
oxaliplatin, and 5-FU. Accordingly, in a first aspect, the present
invention provides a method of treating hepatocellular carcinoma,
which comprises treating a patient identified as having
hepatocellular carcinoma, with a therapeutically effective amount
of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)].
[0007] In a second aspect, the present invention provides a method
of preventing or delaying the onset of hepatocellular carcinoma,
comprising administering to a patient identified to be in need of
prevention, or delaying the onset, of hepatocellular carcinoma a
prophylatically effective amount of sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)].
[0008] The present invention further provides use of sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] for the
manufacture of a medicament useful for treating, preventing or
delaying the onset of hepatocellular carcinoma.
[0009] In yet another aspect, the present invention provides a
method of treating refractory or resistant hepatocellular carcinoma
comprising identifying a patient having refractory hepatocellular
carcinoma and treating the patient with a therapeutically effective
amount of sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)].
In specific embodiments, the patient has a hepatocellular carcinoma
that is refractory to a treatment comprising one or more drugs
selected from the group consisting of sorafenib, regorafenib,
doxorubicin, cisplatin, carboplatin, oxaliplatin, 5-FU and
capecitabine.
[0010] The foregoing and other advantages and features of the
invention, and the manner in which the same are accomplished, will
become more readily apparent upon consideration of the following
detailed description of the invention taken in conjunction with the
accompanying examples, which illustrate preferred and exemplary
embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a graph showing the cytotoxicity of sodium salt of
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] in different
hepatocellular carcinoma cell lines;
[0012] FIG. 2 is a graph showing the activity of sodium salt of
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] in inducing
apoptosis in Hep3B cell line;
[0013] FIG. 3 is a gel image showing that apoptotic cell death
induced by sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]
is further characterized by PARP cleavage;
[0014] FIG. 4 is a plot showing that treatment with sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] was found to
significantly reduce DNA synthesis rate measured by
.sup.3H-thymidine incorporation;
[0015] FIG. 5 is a plot showing the activity of sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] ("Test Drug")
against xenografted hepatocellular carcinoma (from Hep3B cells) in
female SCID mice as compared to treatment with sorafenib;
[0016] FIG. 6 is a plot showing that sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] ("Test Drug") led
to a significant increase in life span of female SCID mice
xenografed with hepatocellular carcinoma formed from Hep3B
cells.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention is at least in part based on the
discovery that the compound sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] is especially
effective in treating hepatocellular carcinoma. Accordingly, in
accordance with a first aspect of the present invention, a method
is provided for treating hepatocellular carcinoma (or malignant
hepatoma). Specifically, the method comprises treating a patient
having hepatocellular carcinoma with a therapeutically effective
amount of trans-[tetrachlorobis(1H-indazole)ruthenate(III)] or a
pharmaceutically acceptable salt thereof, such as an alkali metal
salt of trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (e.g.,
sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] or
potassium trans-[terachlorobis(1H-indazole)ruthenate(III)]) and
indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]. That
is, the present invention is directed to the use of
trans-[terachlorobis(1H-indazole)ruthenate(III)] or a
pharmaceutically acceptable salt thereof (an alkali metal salt or
indazolium salt) for the manufacture of medicaments for treating
hepatocellular carcinoma in patients identified or diagnosed as
having hepatocellular carcinoma.
[0018] In the various embodiments of this aspect of the present
invention, the treatment method optionally also comprises a step of
diagnosing or identifying a patient as having hepatocellular
carcinoma. The identified patient is then treated with or
administered with a therapeutically effective amount of a
pharmaceutically acceptable salt of
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] such as an alkali
metal salt of trans-[tetrachlorobis(1H-indazole)ruthenate(III)]
(e.g., sodium trans-[terachlorobis(1H-indazole)ruthenate(III)] or
potassium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]).
Hepatocellular carcinoma can be diagnosed in any conventional
diagnostic methods known in the art including ultrasound, CT scan,
MRI, alpha-fetoprotein testing, des-gamma carboxyprothrombin
screening, and biopsy.
[0019] In addition, it has also been surprisingly discovered that
the compound sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] is equally
effective in hepatocellular carcinoma cell lines both sensitive and
insensitive to drugs such as sorafenib, doxorubicin, cisplatin,
oxaliplatin, and 5-FU. Accordingly, the present invention also
provides a method of treating refractory hepatocellular carcinoma
or hepatocellular carcinoma comprising treating a patient
identified as having refractory hepatocellular carcinoma with a
therapeutically effective amount of
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] or a
pharmaceutically acceptable salt thereof such as indazolium salt or
an alkali metal salt of
trans-[terachlorobis(1H-indazole)ruthenate(III)] (e.g., sodium
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] or potassium
trans-[tetrachlorobis(1H-indazole)ruthenate(III)]). In specific
embodiments, the patient has a hepatocellular carcinoma that is
refractory to a treatment comprising one or more drugs selected
from the group consisting of sorafenib, regorafenib, anthracyclines
(e.g., doxorubicin, daunorubicin, epirubicin, idarubicin), platinum
agents (e.g., cisplatin, carboplatin, oxaliplatin, picoplatin),
5-FU and capecitabine. That is, the present invention is also
directed to the use of an alkali metal salt of
trans-[terachlorobis(1H-indazole)ruthenate(III)] (e.g., sodium
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] or potassium
trans-[terachlorobis(1H-indazole)ruthenate(III)]) for the
manufacture of medicaments for treating refractory hepatocellular
carcinoma, e.g., a hepatocellular carcinoma refractory to one or
more drugs chosen from sorafenib, regorafenib, anthracyclines
(e.g., doxorubicin, daunorubicin, epirubicin, idarubicin), platinum
agents (cisplatin, carboplatin, oxaliplatin, picoplatin), 5-FU and
capecitabine.
[0020] The term "refractory hepatocellular carcinoma," as used
herein refers to hepatocellular carcinoma that either fails to
respond favorably to an antineoplastic treatment that does not
include trans-[terachlorobis(1H-indazole)ruthenate(III)] or a
pharmaceutically acceptable salt thereof, or alternatively, recurs
or relapses after responding favorably to an antineoplastic
treatment that does not include
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] or a
pharmaceutically acceptable salt thereof. Accordingly, "a
hepatocellular carcinoma refractory to a treatment" as used herein
means a hepatocellular carcinoma that fails to respond favorably
to, or resistant to, the treatment, or alternatively, recurs or
relapses after responding favorably to the treatment.
[0021] Thus, in some embodiments, in the method of the present
invention, sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]
is used to treat hepatocellular carcinoma patients having a tumor
that exhibits resistance to a treatment comprising one or more
drugs selected from the group consisting of sorafenib, regorafenib,
anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin,
idarubicin), platinum agents (e.g., cisplatin, carboplatin,
oxaliplatin, picoplatin), 5-FU and capecitabine. In other words,
the method is used to treat a hepatocellular carcinoma patient
having previously been treated with a treatment regimen that
includes one or more drugs selected from the group consisting of
sorafenib, regorafenib, anthracyclines (e.g., doxorubicin,
daunorubicin, epirubicin, idarubicin), platinum agents (e.g.,
cisplatin, carboplatin, oxaliplatin, picoplatin), 5-FU, tegafur and
capecitabine, and whose hepatocellular carcinoma was found to be
non-responsive to the treatment regimen or have developed
resistance to the treatment regimen. In other embodiments, the
method is used to treat a hepatocellular carcinoma patient
previously treated with a treatment comprising one or more drugs
selected from the group consisting of sorafenib, regorafenib,
anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin,
idarubicin), platinum agents (e.g., cisplatin, carboplatin,
oxaliplatin, picoplatin), 5-FU and capecitabine, but the
hepatocellular carcinoma has recurred or relapsed, that is, a
hepatocellular carcinoma patient who has previously been treated
with one or more such drugs, and whose cancer was initially
responsive to the previously administered one or more such drugs,
but was subsequently found to have relapsed. In specific
embodiments, sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] is used to treat
hepatocellular carcinoma patients previously treated with sorafenib
or regorafenib, i.e., who have a tumor that exhibits resistance to,
or relapsed after a treatment including, sorafenib or regorafenib.
In other specific embodiments, sodium
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is used to treat
hepatocellular carcinoma patients previously treated with
doxorubicin, i.e., who have a hepatocellular carcinoma that
exhibits resistance to, or relapsed after a treatment including,
doxorubicin. In yet other specific embodiments, sodium
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is used to treat
hepatocellular carcinoma patients previously treated with a
platinum cytotoxic agent (e.g., cisplatin, carboplatin,
oxaliplatin, picoplatin), i.e., who have a hepatocellular carcinoma
that exhibits resistance to, or relapsed after a treatment
including, a platinum cytotoxic agent (e.g., cisplatin,
carboplatin, picoplatin, oxaliplatin, or picoplatin). In still
other specific embodiments, sodium
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is used to treat
hepatocellular carcinoma patients previously treated with 5-FU or a
prodrug thereof (e.g., tegafur or capecitabine or S1), i.e., who
have a hepatocellular carcinoma that exhibits resistance to, or
relapsed after a treatment including, 5-FU or or a prodrug thereof
(e.g., tegafur or capecitabine or S1).
[0022] To detect a refractory hepatocellular carcinoma, patients
undergoing initial treatment can be carefully monitored for signs
of resistance, non-responsiveness or recurring hepatocellular
carcinoma. This can be accomplished by monitoring the patient's
cancer's response to the initial treatment which, e.g., may
includes one or more drugs selected from the group consisting of
sorafenib, regorafenib, doxorubicin, daunorubicin, epirubicin,
idarubicin, cisplatin, carboplatin, oxaliplatin, picoplatin, 5-FU,
tegafur and capecitabine. The response, lack of response, or
relapse of the cancer to the initial treatment can be determined by
any suitable method practiced in the art. For example, this can be
accomplished by the assessment of tumor size and number. An
increase in tumor size or, alternatively, tumor number, indicates
that the tumor is not responding to the chemotherapy, or that a
relapse has occurred. The determination can be done according to
the "RECIST" criteria as described in detail in Therasse et al, J.
Natl. Cancer Inst. 92:205-216 (2000).
[0023] In accordance with yet another aspect of the present
invention, a method is provided for preventing or delaying the
onset of hepatocellular carcinoma (or hepatocellular carcinoma), or
preventing or delaying the recurrence of hepatocellular carcinoma,
which comprises treating a patient in need of the prevention or
delay with a prophylatically effective amount of a pharmaceutically
acceptable salt of
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] such as an alkali
metal salt of trans-[tetrachlorobis(1H-indazole)ruthenate(III)]
(e.g., sodium trans-[terachlorobis(1H-indazole)ruthenate(III)] or
potassium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]).
[0024] It is now known that people with hepatitis B or hepatitis C
infection, or having cirrhosis have an increased risk of developing
hepatocellular carcinoma. In addition, people who have acute and
chronic hepatic porphyrias (acute intermittent porphyria, porphyria
cutanea tarda, hereditary coproporphyria, variegate porphyria) and
tyrosinemia type I are also at an increased risk of for developing
hepatocellular carcinoma. These people can all be candidates for
the method of present invention for preventing or delaying the
onset of hepatocellular carcinoma using a prophylatically effective
amount of, a pharmaceutically acceptable salt of
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] such as an alkali
metal salt of trans-[terachlorobis(1H-indazole)ruthenate(III)]
(e.g., sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] or
potassium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]). In
addition, patients with a family history of hepatocellular
carcinoma can also be identified for the application of the present
method of preventing or delaying the onset of hepatocellular
carcinoma.
[0025] For purposes of preventing or delaying the recurrence of
hepatocellular carcinoma, hepatocellular carcinoma patients who
have been treated and are in remission or in a stable or
progression free state may be treated with a prophylatically
effective amount of a pharmaceutically acceptable salt of
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] such as an alkali
metal salt of trans-[tetrachlorobis(1H-indazole)ruthenate(III)]
(e.g., sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] or
potassium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]) to
effectively prevent or delay the recurrence or relapse of
hepatocellular carcinoma.
[0026] As used herein, the phrase "treating . . . with . . . " or a
paraphrase thereof means administering a compound to the patient or
causing the formation of a compound inside the body of the
patient.
[0027] In accordance with the method of the present invention,
hepatocellular carcinoma can be treated with a therapeutically
effective amount of a pharmaceutically acceptable salt of
trans-[terachlorobis(1H-indazole)ruthenate(III)] such as an alkali
metal salt of trans-[tetrachlorobis(1H-indazole)ruthenate(III)]
(e.g., sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] or
potassium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]) alone
as a single agent, or alternatively in combination with one or more
other anti-cancer agents.
[0028] Alkali metal salts of
trans-[terachlorobis(1H-indazole)ruthenate(III)] can be made in any
methods known in the art. For example, PCT Publication No.
WO/2008/154553 discloses an efficient method of making sodium
trans-[tetrachlorobis(1H-indazole)ruthenate(III)].
[0029] The pharmaceutical compounds such as sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] can be
administered through intravenous injection or any other suitable
means at an amount of from 0.1 mg to 1000 mg per kg of body weight
of the patient based on total body weight. The active ingredients
may be administered at once, or may be divided into a number of
smaller doses to be administered at predetermined intervals of
time, e.g., once daily or once every two days. It should be
understood that the dosage ranges set forth above are exemplary
only and are not intended to limit the scope of this invention. The
therapeutically effective amount of the active compound can vary
with factors including, but not limited to, the activity of the
compound used, stability of the active compound in the patient's
body, the severity of the conditions to be alleviated, the total
weight of the patient treated, the route of administration, the
ease of absorption, distribution, and excretion of the active
compound by the body, the age and sensitivity of the patient to be
treated, and the like, as will be apparent to a skilled artisan.
The amount of administration can be adjusted as the various factors
change over time.
[0030] In accordance with the present invention, it is provided a
use of a pharmaceutically acceptable salt of
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] such as an alkali
metal salt of trans-[tetrachlorobis(1H-indazole)ruthenate(III)]
(e.g., sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] or
potassium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]) for
the manufacture of a medicament useful for treating hepatocellular
carcinoma. The medicament can be, e.g., in an injectable form,
e.g., suitable for intravenous, intra-arterial, intradermal, or
intramuscular administration. Injectable forms are generally known
in the art, e.g., in buffered solution or suspension.
[0031] In addition, a pharmaceutically acceptable salt of
trans-[terachlorobis(1H-indazole)ruthenate(III)] such as an alkali
metal salt of trans-[terachlorobis(1H-indazole)ruthenate(III)] can
also be used in chemoembolization, in which the drug is
administered directly into tumor, while an embolizing agent is used
to block the blood supply to the tumor thereby trapping the drug
within the tumor. For example, lipiodol (iodized oil) and
.sup.131I-lipidol are embolizing agents suitable for use with an
alkali metal salt of
trans-[terachlorobis(1H-indazole)ruthenate(III)] in
chemoembolization. Other useful embolizing agents include gelatin
(e.g. GelFoam) or degradable starch microspheres of defined size
ranges. These embolizing agents can be given via intrahepatic
artery along with an alkali metal salt of
trans-[terachlorobis(1H-indazole)ruthenate(III)] via the same
route, known as "hepatic artery transcatheter treatments."
[0032] Thus, the method of the present invention also provides a
method for treating hepatocellular carcinoma, preventing or
delaying the onset of refractory or recurrent hepatocellular
carcinoma comprising administering to a patient a pharmaceutically
acceptable salt of trans-[terachlorobis(1H-indazole)ruthenate(III)]
such as an alkali metal salt of
trans-[terachlorobis(1H-indazole)ruthenate(III)] and an embolizing
agent. The alkali metal salt of
trans-[terachlorobis(1H-indazole)ruthenate(III)] and embolizing
agent such as lipiodol (iodized oil), .sup.131I-lipidol and gelatin
can be administered by hepatic artery injection or intraarterial
infusion. Administration of lipiodol or .sup.131I-lipidol for
treating hepatocellular carcinoma is generally known in the
art.
[0033] In accordance with another aspect of the present invention,
a pharmaceutical kit is provided comprising in a container a unit
dosage form of a pharmaceutically acceptable salt of
trans-[terachlorobis(1H-indazole)ruthenate(III)] such as an alkali
metal salt of trans-[tetrachlorobis(1H-indazole)ruthenate(III)]
(e.g., sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] or
potassium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]), and
optionally instructions for using the kit in the methods in
accordance with the present invention, e.g., treating, preventing
or delaying the onset of hepatocellular carcinoma, or preventing or
delaying the recurrence of hepatocellular carcinoma, or treating
refractory hepatocellular carcinoma. As will be apparent to a
skilled artisan, the amount of a therapeutic compound in the unit
dosage form is determined by the dosage to be used on a patient in
the methods of the present invention. In the kit, a
pharmaceutically acceptable salt of
trans-[terachlorobis(1H-indazole)ruthenate(III)] such as an alkali
metal salt of trans-[tetrachlorobis(1H-indazole)ruthenate(III)]
(e.g., sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] or
potassium trans-[terachlorobis(1H-indazole)ruthenate(III)]) can be
in lyophilized form in an amount of, e.g., 25 mg, in an ampoule. In
the clinic, the lyophilized form can be dissolved and administered
to a patient in need of the treatment in accordance with the
present invention. In addition, the kit optionally further includes
a unit dose form of an embolizing agent such as lipiodol (iodized
oil) or .sup.131I-lipidol, which can be, e.g., in vials each having
1 to 1000 ml of lipiodol (iodized oil) or .sup.131I-lipidol or
gelatine or microspheres. In addition, the present invention also
provides a pharmaceutical composition comprising a therapeutically
effective amount of a pharmaceutically acceptable salt of
trans-[terachlorobis(1H-indazole)ruthenate(III)] such as an alkali
metal salt of trans-[terachlorobis(1H-indazole)ruthenate(III)]
(e.g., sodium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] or
potassium trans-[terachlorobis(1H-indazole)ruthenate(III)]) in
admixture with an embolizing agent (e.g., lipiodol (iodized oil) or
.sup.131I-lipidol, or gelatine).
EXAMPLE
[0034] To test the activities of sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] ("drug"), MTT
assays were performed using selected hepatocellular carcinoma cell
lines. Cells were plated (2.times.10.sup.3 cells in 100 .mu.l/well)
in 96-well plates and allowed to recover for 24 hours. The drug was
added in another 100 .mu.l growth medium and incubated with
cultured cells for 3 hours before the cell culture medium was
replaced to remove the drug. Cell death was measured 72 hours after
the initial incubation by MTT assay following the manufacturer's
recommendations (EZ4U, Biomedica, Vienna, Austria). After 72 hours
of treatment, the mean IC.sub.50 value was 124.4 .mu.M with HCC1.2,
and HCC3 being most sensitive (IC.sub.50 values of 62.9 .mu.M and
67.5 .mu.M, respectively) (FIG. 1). Notably, the IC.sub.50 values
did not correlate with intracellular drug levels determined by
inductively-coupled plasma mass spectrometry (ICP-MS) measurements.
All tested cell lines revealed similar Ru contents (.about.5 ng
Ru/105 cells) after 1 hour drug exposure.
[0035] To evaluate the mechanisms underlying the drug activity,
apoptosis induction as well as impact on cell cycle distribution
and proliferation of Hep3B cells were evaluated (FIG. 2).
Specifically, the hepatoma cell line Hep3B was purchased from
American Type Culture Collection (ATCC), Manassas, Va. All cells
were grown in RPMI 1640 supplemented with 10% FCS. Cultures were
regularly checked for Mycoplasma contamination. After 24 hours of
drug treatment, cells were harvested, washed in PBS and cytospins
were prepared. After fixation with a 1:1 methanol/acetone solution,
slides were stained with 4',6-diamidino-2-phenylindole (DAPI)
containing antifade solution (Vector Laboratories, Inc., USA).
Nuclear morphology of cells was examined using a Laica DMRXA
fluorescence microscope (Laica Mikroskopie and System, Wetzlar,
Germany) equipped with appropriate epifluorescence filters and a
COHU charge-douples device camera. Duplicate slides were prepared
for each cell type/treatment group and 300-500 cells were counted
for each sample. Sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] led to significant
apoptosis in Hep3B cells.
[0036] Treatment with sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] led to typical
morphological signs of programmed cell death like cell shrinkage,
chromatin condensation and formation of apoptotic bodies. Apoptotic
cell death induced by sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] was further
characterized by PARP cleavage examined by Western Blot Analyses.
Specifically, after 24 hours of drug treatment, protein extracts
were prepared and Western blot analyses performed. Rabbit Anti-PARP
from Apoptosis Sampler kit (Cell Signaling Technology, Beverly,
Mass.) (dilution 1:1000)000) was used. For loading control
.beta.-actin monoclonal mouse AC-15 (Sigma, USA, dilution: 1:1000)
was used. All secondary peroxidase-labeled antibodies were
purchased from Santa Cruz Biotechnology and used at working
dilution of 1:10000. FIG. 3 shows PARP cleavage induced by sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)].
[0037] An increase of cells with mitochondrial membrane
depolarization (measured by JC-1 staining and FACS analysis)
suggested that at least part of the observed apoptotic cell death
is induced via the mitochondrial pathway. Furthermore, treatment
with sodium trans-[terachlorobis(1H-indazole)ruthenate(III)] was
found to significantly reduce DNA synthesis rate measured by
.sup.3H-thymidine incorporation (FIG. 4).
[0038] With regard to cell cycle distribution, 24 hours of
treatment led to increase of cells in G2/M phase (detected by
PI-staining followed by FACS analyses). This was accompanied by
strong phosphorylation of the stress kinase P38 and the
extracellular signal-regulated kinase (ERK), suggesting that P38
might be involved in delayed apoptosis entry.
[0039] Separately, the activity of sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] against Hep3B
cells was evaluated in xenograft experiments using female SCID mice
as compared to the standard therapy sorafenib. Sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] was administered W
at 30 mg/kg once a week for two weeks. Sorafenib was administered
P.O. at 25 mg/kg daily five times a week for two weeks. Sodium
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] was well
tolerated. In the Hep3B model, sodium
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] effectively
suppressed the growth of tumor (FIG. 5).
[0040] In addition, sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] led to a 2.4-fold
increase in life span (mean survival of 80 days as compared to 33
days in control group) and thus was superior to sorafenib
monotherapy (1.9-fold survival increase; 60 days as compared to 33
days in the control) (FIG. 6). Taken together, sodium
trans-[tetrachlorobis(1H-indazole)ruthenate(III)] is a very
promising anticancer drug with significant activity against human
hepatocellular carcinoma cell lines in vitro and in vivo.
[0041] To determine whether the cytotoxicity of sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] varies in
hepatocellular carcinoma cells sensitive and insensitive (or
significantly less sensitive) to other drugs, the IC.sub.50 values
of sorafenib, doxorubicin, and oxaliplatin in various
hepatocellular carcinoma cell lines were also determined. In
addition, the IC.sub.50 values of 5-FU and cisplatin in the Hep3B
and HepG2 cell lines were obtained from Kogure et al., Cancer
Chemother. Pharmacol., 53(4):296-304 (2004). The ratios of
IC.sub.50 values of sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] in a cell line
sensitive to one of the other drugs and a cell line insensitive to
the same drug were calculated. The results are shown in Tables 1-5
below ("Test Drug" in the tables denotes sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)]). The data shows
that sodium trans-[terachlorobis(1H-indazole)ruthenate(III)] is
equally effective in hepatocellular carcinoma cells that are
sensitive or resistant to other drugs such as sorafenib,
doxorubicin, 5-FU, and platinum agents such as oxaliplatin, and
cisplatin. As such, sodium
trans-[terachlorobis(1H-indazole)ruthenate(III)] is potentially
effective in treating hepatocellular carcinoma resistant to such
other drugs.
TABLE-US-00001 TABLE 1 Test Drug Sorafenib HCC IC.sub.50 Ratio
IC.sub.50 Ratio Cell Line IC.sub.50 (HCC3/B1) IC.sub.50 (HCC3/B1)
B1 83.4 .mu.M 0.8 0.8 .mu.M >12.5 HCC3 67.5 .mu.M >10
.mu.M
TABLE-US-00002 TABLE 2 Test Drug Doxorubicin HCC IC.sub.50 Ratio
IC.sub.50 Ratio Cell Line IC.sub.50 (HepG2/Hep3B) IC.sub.50
(HepG2/Hep3B) Hep3B 166.9 .mu.M 0.8 69.25 .mu.M >3.6 HepG2 188.8
.mu.M >250 .mu.M
TABLE-US-00003 TABLE 3 Test Drug Oxaliplatin HCC IC.sub.50 Ratio
IC.sub.50 Ratio Cell Line IC.sub.50 (HCC2/HCC3) IC.sub.50
(HCC2/HCC3) HCC2 128.9 .mu.M 1.9 4.05 .mu.M >9.9 HCC3 67.5 .mu.M
0.41 .mu.M
TABLE-US-00004 TABLE 4 Test Drug Cisplatin HCC IC.sub.50 Ratio
IC.sub.50 Ratio Cell Line IC.sub.50 (HepG2/ Hep3B) IC.sub.50
(HepG2/Hep3B) Hep3B 166.9 .mu.M 1.1 3.75 .mu.g/ml >5.2 HepG2
188.8 .mu.M 19.4 .mu.g/ml
TABLE-US-00005 TABLE 4 Test Drug 5-FU HCC IC.sub.50 Ratio IC.sub.50
Ratio Cell Line IC.sub.50 (HepG2/ Hep3B) IC.sub.50 (HepG2/Hep3B)
Hep3B 166.9 .mu.M 1.1 227 .mu.g/ml >5.2 HepG2 188.8 .mu.M 17.2
.mu.g/ml
[0042] All publications and patent applications mentioned in the
specification are indicative of the level of those skilled in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference. The mere mentioning of the publications and patent
applications does not necessarily constitute an admission that they
are prior art to the instant application.
[0043] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be apparent that certain changes and
modifications may be practiced within the scope of the appended
claims.
* * * * *